S-layer O-glycans were isolated from T. forsythia mutant cells following a published protocol (Posch et al., 2011 (link)). Briefly, S-layer glycoproteins were excised from Coomassie Brilliant Blue R-250-stained SDS-PA gels and O-glycans were released from the protein backbone by in-gel reductive β-elimination followed by removal of excess of salt using a 25-mg HyperSep Hypercarb SPE cartridge (Thermo Scientific). Borohydride-reduced O-glycans were analyzed by PGC-ESI-MS/MS as described recently (Hypercarb, 0.32 × 150 mm, particle size 5 μm) (Stadlmann et al., 2008 (link)). Detection was done by an ESI-Q-TOF Global Ultima from Micromass (Waters, USA). Data were evaluated using MassLynx 4.0 software. MS/MS experiments were performed at 30% collision energy using CID with argon gas. Slices from the SDS-polyacrylamide gel without sample application were treated in the same way as described for the O-glycan samples and acquired MS spectra were used as a negative control.